Cell shape controls rheotaxis in small parasitic bacteria

Mycoplasmas, a group of small parasitic bacteria, adhere to and move across host cell surfaces. The role of motility across host cell surfaces in pathogenesis remains unclear. Here, we used optical microscopy to visualize rheotactic behavior in three phylogenetically distant species of Mycoplasma using a microfluidic chamber that enabled the application of precisely controlled fluid flow. We show that directional movements against fluid flow occur synchronously with the polarized cell orienting itself to be parallel against the direction of flow. Analysis of depolarized cells revealed that morphology itself functions as a sensor to recognize rheological properties that mimic those found on host-cell surfaces. These results demonstrate the vital role of cell morphology and motility in responding to mechanical forces encountered in the native environment.


Introduction
Mycoplasmas are small parasitic bacteria with small genomes that lack a cell wall [1]. These relatively simple and small species lack the surface appendages important for host colonization found in other bacterial pathogens, such as flagella or pili. Some species of Mycoplasma, however, exhibit a type of motility known as gliding motility [2,3]. Currently, over ten Mycoplasma axis was aligned parallel with the direction of flow ( Fig 1C). Hereafter, we refer to upstreamdirected motility as positive rheotaxis. Positive rheotactic behavior was observed for over 80% of gliding M. pneumoniae cells (N = 50 cells). In our experimental setup, we observed cells at 30˚C and not at the optimal growth temperature of 37˚C. This lower temperature minimized thermal drift during the acquisition of cell images. Although the gliding speed decreased to 0.18 ± 0.04 μm/s at 30˚C, which is 50% slower than that at 37˚C, the cells still showed clear and smooth gliding motility (S2 Fig). All experiments with M. pneumoniae were performed at 30˚C unless otherwise stated.
We next examined the dependency of cell movement on flow speed to gain further insight into positive rheotactic behavior by M. pneumoniae (Fig 2A). We found that at flow speeds greater than 0.5 mm/s, cells changed their previously random orientations to adopt orientations parallel to the direction of flow. During positive rheotaxis, the angle of the moving  direction relative to the flow axis was 0˚, on average. The cell trajectories of rheotaxing cells were observed to be straight with no bias, while that those in no-flow conditions have been reported to be biased to the right (clockwise when viewed from above) [29]. In agreement with previous observations, we found that the mean square displacement (MSD) of M129 cells increased linearly over time in the absence of flow, demonstrating that gliding motility sans flow is random with an apparent diffusion coefficient of 0.2 μm 2 /s (Fig 2B, Blue). In contrast, the MSD of cells at a flow speed of 1.2 μm/s had a parabolic line, indicating that gliding motility is directional under flow (Fig 2B, Red).
Positive rheotactic behavior was found to stop at flow speeds >4.0 mm/s, but orientation of the long axis of the cell parallel to the direction of flow persisted (Fig 2C). These stationary cells did not detach even at 15 mm/s, the fastest flow speed in our experimental setup. Some cells under flow were categorized as having different motility patterns than the majority of cells, such as detachment from the surface or being dragged backward, but the proportion of these two motility patterns combined was less than 20% of the cell population. We report cell displacement in the direction of flow as negative values in our measurements.
Positive-rheotactic gliding velocity showed a positive correlation as a function of the flow speed over the range of 0.5-3.8 mm/s, which was linearly extrapolated to zero to give a flow speed of 4.1 mm/s ( Fig 2D). Clinical isolates of M. pneumoniae can be classified into two major groups, with strain M129 a representative of type 1 isolates, and another strain, FH, representative of type 2 isolates [30]. The positive correlation between the flow speed and cell velocity during positive rheotaxis was also observed in FH (Fig 2D and S2 Movie).
To determine whether there is a link between rheotaxis and gliding motility, we observed the behavior of mutants of M. pneumoniae M129 possessing AOs of different sizes (S3 Fig). The dec_5 and inc_5 mutants, harboring size-modified HMW2 derivatives, form longer and shorter AOs, respectively [31]. We found that in both of these backgrounds, cells showed positive rheotactic behavior. Gliding velocity showed a positive correlation as a function of flow speed over a range of 0.8-1.7 mm/s. However, extrapolation of gliding speed at zero to the flow velocity was slower than that from WT (S3 and 2D Figs). We speculate that this could be caused by impaired binding of the attachment organelle to the glass surface in size-modified HMW2 mutants [31].
To determine whether M. pneumoniae can counter the force of airway-cilia-generated mucus flow, we also analyzed rheotaxis in viscous environments ( Fig 3A and S3 Movie). We added methylcellulose (MC) at concentrations of 0.25%-0.5% to increase viscosity of the fluid in the flow chamber. We found that viscous-fluid flow had a large effect on the gliding-motility speed of cells, relative to non-viscous-fluid flow. Half of the cell population was stationary at a flow speed of 1.0 mm/s, and most cells detached from the glass surface at a flow speed of 3.2 mm/s in the presence of 0.5% MC (Fig 3B and 3C and S4 Movie).

Cell shape affects Rheotaxis
Over the course of our experiments, we often observed small cellular particles exhibiting gliding motility in the flow chamber. These particles exhibited random gliding motility under flow (i.e. rheotaxis was not observed) (S5 Movie). Intact cells adhere to the glass surface at the leading end of the cell where the AO is localized. The small cellular particles we observed appeared to be composed of a cell fragment containing the AO, but lacking the rest of the cell body ( Fig  4A). Immunofluorescent microscopy against the P1 protein, a major surface component of the AO (Fig 4B) and transmission electron microscopy (TEM) confirmed that the small cellular particle was the leading end of the cell that had detached from the rest of the cell body ( Fig  4C). Previous work has reported that some mutants of M. pneumoniae produce isolated AOs, which lack DNA and continue to glide for up to 30 minutes after separating from an intact cell [32].
To study gliding motility and rheotaxis of detached but otherwise WT AOs, we applied shear force to a WT cell suspension, which produced numerous detached AOs for observation. The presence of isolated AOs was confirmed by their small size and smooth movement over the glass surface, as well as a gliding velocity~1/20 th that of the intact cells. The isolated AOs exhibited gliding motility with curved trajectories even at a flow speed of 1.7 mm/s ( Fig 4D) and a reduced dependence on flow speed to stimulate rheotaxis compared to intact cells ( Fig  4E). This suggests that polar cell shape and asymmetric cell binding are required for recognition of flow direction during rheotaxis ( Fig 4F).

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Cell shape controls rheotaxis in small parasitic bacteria

Rheotaxis in M. mobile and M. penetrans
We next examined the relationship between motility and flow speed in M. mobile and M. penetrans, phylogenetically distant species from M. pneumoniae, in order to generalize our findings ( Fig 5A) [4,10]. We found that M. mobile showed curved trajectories without flow in the sample chamber, as previously reported [29], but that cells assumed straight, upstream trajectories at a flow speed of 1 mm/s ( Fig 5B upper and S6 Movie). In contrast, M. penetrans did not attach to the glass surface under the same conditions, which is consistent with a previous study which reported that gliding motility by M. penetrans requires supplementation of the media with 3% gelatin [7]. To mimic the addition of gelatin, we increased the fluid viscosity by adding 0.5% MC and found that M. penetrans exhibited smooth gliding motility under these conditions (S7  (S4B Fig), where the terminal organelle has been suggested to be localized (S4C Fig) [7]. These curved trajectories changed to straight when we applied a fluid flow of 0.6 mm/ s (Fig 5B lower and S8 Movie). We observed a motility pattern by M. penetrans that shifted from random under no flow, to positive rheotaxis, to stationary, and finally to detachment from the glass surface as the flow speed increased (Fig 5C lower).
Unlike M. pneumoniae or M. penetrans, cells of M. mobile did not stop gliding motility at higher flow speeds (Fig 5C upper). We plotted the velocity of gliding motility as a function of the flow speed measured at a temperature of 25˚C. This revealed a positive correlation, which was linearly extrapolated to zero-gliding velocity to give flow speeds of 8.4 mm/s and 1.3 mm/s in M. mobile and M. penetrans, respectively (Fig 5D).

Discussion
In this report, we directly visualized positive rheotaxis by M. pneumoniae and revealed that the directionality of rheotaxis is controlled by polar cell shape (Figs 1, 2, and 4). This is consistent with previous work which has shown that upstream motion in other microorganisms is promoted by asymmetric cell shape or asymmetric binding [20,22,33]. By closely examining cell movement in M. pneumoniae, we found that, under fluid flow, the rear part of the cell body rotates around the attachment organelle, enabling the long axis of the cell to align parallel to the flow axis to minimize fluid resistance. This alignment appears to be a simple mechanical response where the cell body acts as an antenna to recognize flow direction, similar to a weathervane (Fig 4F).
Polar cell shape and asymmetric cell binding are conserved in almost all gliding mycoplasmas [5,7,9,34], suggesting that this framework of passive control of rheotaxis also applies to phylogenetically distant species, such as M. mobile and M. penetrans (Fig 5E) [11,12,35]. As the molecular basis of gliding motility has no obvious similarity among these species, unidirectional gliding motility may have arisen via convergent evolution to enable rheotactic behavior in response to external mechanical signals.
M. pneumoniae cells exhibited rheotaxis in highly viscous environments at flow speeds up to 3.2 mm/s (Fig 3). By approximating cell shape as a cylinder, we estimated the maximum force exerted during gliding motility to be 150 pN (S5 Fig), which is higher than the stall force in M. mobile [28,36]. Thus, the gliding force of a single M. pneumoniae cell is sufficient to counter that of human airway cilia at 60 pN [37], indicating that cells are not swept away by mucociliary clearance.
Many biological interactions are enhanced by load-dependent mechanisms called catch bonds, which have been well-studied in bacteria [38], and have recently also been suggested to exist in M. mobile [39]. The adherence of M. pneumoniae and M. mobile is postulated to involve surface proteins [40,41] that bind sialic acid targets on the host cells [42]. These surface proteins may possess rheological properties in the actual host-surface environment.
We hypothesized that rheotaxis might play a critical role for pathogenicity in gliding mycoplasmas. Once M. pneumoniae cells have been transmitted to the host airway through aerosols [13], the unidirectional flow of the mucociliary defense system of the host enables M. pneumoniae to navigate to the lungs, opposite the direction of mucociliary flow. Here, we showed that cells exhibit rheotaxis even at a flow speed of 1.0 mm/s in the presence of 0.5% MC (Fig 3). This experimental flow speed is much greater than that of in vivo mucociliary transport in a newborn pig model system, which was reported as 0.1 mm/s [43].

Cell shape controls rheotaxis in small parasitic bacteria
The flow-induced positive rheotaxis we observed in this study might be applicable to other gliding mycoplasmas. M. mobile was isolated from the gills of a tench (Tinaca tinca) [44], around whose gills the fluid flow speed was reported to be 7.6 mm/s [45], which has good agreement with our measurement of the maximum flow speed at which M. mobile remained adhered in the flow chamber (Fig 5). M. penetrans was originally isolated from the urinary tract [46], where ureter peristalsis drives the flow of urine from the kidneys to the bladder at speeds that reach approximately 20-30 mm/s [47]. Our measurement suggests that M. penetrans cells can adhere at a flow speed of 36 mm/s in low-viscosity fluids such as urine (Fig 5). Additionally, the maximum force exerted during gliding motility by M. penetrans is higher than that of type I pili in uropathogenic Escherichia coli at 60 pN, suggesting that the force of gliding motility in M. penetrans is sufficient to persist in the urinary tract [48].
The measurements in this study were taken from experiments in a simplified flow chamber, where the temperature is controllable up to 30˚C. The flow inside the chamber is typically laminar, and flow speed decreases at the boundary of solid/liquid interface [49]. Consequently, further studies are required to verify our observations of positive rheotaxis in a more realistic environment, e.g., through the use of microfluidic devices with complex shapes or surfaces that resemble the cilia of host cells. Nevertheless, the positive rheotactic behavior observed here sheds light on the physiological relevance of cell motility in a minimal life form.

Cell preparation for gliding motility
M. pneumoniae cells were prepared under optimized conditions for gliding motility, as described previously [31]. The tissue culture flask where M. pneumoniae cells were grown on the bottom was washed twice with PBS/HS, which was composed of 10% horse serum (HS) (Gibco) in phosphate-buffered saline (PBS) of 75 mM sodium phosphate (pH 7.4) and 68 mM NaCl. The cells that remained attached to the bottom of the tissue culture flask were scraped into PBS/HS. The HS used in the preparation of the flow chamber was used without heat inactivation [6,31]. The cell suspension was passed through a syringe needle > three times (21G × 38 mm; Terumo) and filtered with a syringe-driven filter unit (Millex LH 0.45 μm; Millipore) [53,54]. The syringe passaged and filtered cell suspension was subsequently used for flow experiments. For the preparation of the isolated AO, the suspension was passed through the syringe needle >15 times and filtered with a syringe-driven filter unit.
For observation of M. pneumoniae in a viscous environment, cells were scraped into either 0.25% or 0.5% MC (M0512, 4,000 cP at 2% solution; Sigma-Aldrich) with 10% HS in PBS and used for flow experiments.
For observation of M. mobile, the cell suspensions were centrifuged at 12,000 × g for 4 min, and the pellet was suspended in PBS/HS at one-tenth of the original culture volume, and subsequently used for flow experiments. For M. penetrans, the cell suspension was centrifuged at 12,000 × g for 4 min. The resulting pellet was suspended in PBS/HS containing 10% fetal bovine serum and 0.5% MC in PBS at one-tenth of the original culture volume and used for flow experiments.

Optical microscopy and data analyses
All measurements were performed under an inverted microscope (IX71; Olympus) equipped with an objective lens (LUCPLFLN 40×PH, N.A. 0.6, LUCPLFLN 60×PH, N.A. 0.7 and UPLANSAPO 100×PH, N.A. 1.4; Olympus), a CMOS camera (Zyla 4.2; Andor, DMK33U174; Imaging Source, and FASTCAM Mini AX; Photoron), and an optical table (HAX-0806; JVI). The microscope was kept at 30˚C by heating the room for the observation of M. pneumoniae cells, but this heating was not applied for M. penetrans and M. mobile. The microscope stage was heated with a thermoplate (TP-110R-100; Tokai Hit) for examining the temperature dependency of gliding speed. Projection of the image to the camera was acquired with imaging software that came with the CMOS camera and converted into a sequential TIFF file without any compression. All data were analyzed by ImageJ v1.48 (rsb.info.nih.gov/ij) [55] and its plugins particle tracker [56] and multitracker.

Flow experiments
The flow chamber was assembled by taping a coverslip with a glass slide, as described previously [57,58]. Inlet and outlet ports were created by boring through the glass slide with a highspeed precision drill press equipped with a diamond-tipped bit [2.35-mm diameter, ICHINEN MTM, Japan]. The sample chamber was prepared from a glass slide, a coverslip (Matsunami), and double-sided tape (*100-μm-thick; 3M). After assembly, the flow chamber was incubated for 60 min at 120˚C to ensure tight adhesion of the slide and coverslip. Inlet and outlet ports (N-333; IDEX Health & Science) were attached with hot-melt adhesive (Goot; Taiyo Electronic IND). The total volume of the sample chambers was adjusted to 10 μL (width: 3 mm, length: 30 mm). A syringe pump (Legato 210P; Kd Scientific) was connected to the flow chamber by a connecter and tube (F-333NX and 1512L; IDEX Health & Science) and used to control the flow rate of the buffer. The flow chamber was pre-coated with PBS/HS, as required. Cell suspensions were pipetted into the chamber and left to incubate for 10 min to allow cells to bind to the glass surface. After washing with PBS/HS or PBS/FBS, the flow chamber was used for observing gliding motility. The flow rate in the sample chamber was calculated by measuring the speed of unattached cells using a high-speed camera (FASTCAM Mini AX; Photoron), as described in the optical microscopy section.

Force estimation
For the drag coefficient of a cell as a cylindrical rod, γ = 2πηL × [ln(L/2r) − 0.20] −1 was applied, where r is cell radius, L is cell length, and η is viscosity (S5 Fig) [59,60]. The η used was 10 −2 and 10 −3 Pa�s for medium with and without 0.5% MC, respectively. The force of gliding motility was estimated by F = γ × v, where v is the gliding speed measured by cell displacement upstream of the fluid flow. The length and the diameter of cells were taken from reported EM images [10,31,63]. The equation for force estimation is described [59,60].

Electron microscopy
Sample preparation for negative-stain EM followed the same protocol described previously [31]. Carbon-coated EM grids were glow-discharged by a PIB-10 hydrophilic treatment device (Vacuum Device). The cell suspension was placed on the EM grid and chemically fixed by 1% (w/v) glutaraldehyde in PBS for 10 min at room temperature. After washing three times with

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Cell shape controls rheotaxis in small parasitic bacteria PBS, the grid was stained with 2% ammonium molybdate (v/v). Samples were observed under a transmission electron microscope (JEM-1400, JEOL) at 100 kV. A charge-coupled device camera captured EM images.